The present invention relates to an autofocus device for a microscope which detects a focal point on a specimen and automatically effects focusing when high resolution observation or fluorescence observation is conducted.
An autofocus (AF) technique has in recent years been adopted so that optimal focus can be realized in a simple manner when a specimen is observed by a microscope. As a device for autofocusing, various techniques have been proposed and of the techniques, a so-called passive method is named in which focusing is effected based on a degree of blurring of an obtained observation image which is viewed (an image including a specimen in a visual field).
For example, a technique according to the Jpn. Pat. Appln. KOKAI Publication No. 9-189850 is that beams are irradiated to a specimen and two optical images of the specimen are formed with transmitted beams in different planes respectively displaced forward and backward relative to an estimated focal plane, contrast levels of the two optical images are obtained, a correct focal position on the specimen is determined through comparison between the contrast levels and then either an objective or a stage on which the specimen is placed is moved to assume an in-focus position and focusing is automatically effected for observation. When a picture is taken, an exposure time is calculated based on a light intensity from the specimen optical image present in an in-focus position and the specimen optical image is shot.
In specimen observation by a microscope, high resolution is first of all required and observation of an image formed by emitted fluorescent light from a specimen has had an increasing chance to be used as an inspection method whose information obtained is rich in volume.
When specimen observation is conducted by a microscope, a scope to be observed in the same visual field at a time is mainly determined by the magnifying power of an objective. In addition, a scope in which an image pick-up device which is mounted on the microscope can take a picture is limited to the visual field.
Generally, in order to obtain an observation image with high resolution, an objective with a high NA and a high magnifying power is mounted, but at that time a visual field is reduced to a very small part of a specimen. With an ordinary microscope, it has been impossible to observe the entire specimen or obtain an image thereof with a broad visual field and high resolution in the same visual field.
On the other hand, for example, a technique whereby an image with a broad visual field and high. resolution are realized is proposed by Jpn. Pat. Appln. KOKAI Publication No. 5-313071.
In this technique, as shown in FIG. 11, a desired visual field is split into a plurality of small areal images, input information is obtained from each small areal image and when the image of a specimen is displayed or printed, the entire visual field is reconstructed as one image and thereby an image of the entire specimen with a broad visual field and high resolution can be achieved.
A problem described below arises when a conventional autofocus technique is adopted as it is in this technique.
In this case, when a desired visual field for observation and shooting is slit into a plurality of small areal images and the shooting is then performed, the shooting is continuously conducted while autofocusing is performed for each small area obtained by the splitting.
That is, as shown in FIG. 11, an observation image is split into nine areas of from X1Y1 to X9Y9 and in shooting, autofocusing is performed for each small areal image.
In this case, since sufficient contrast levels are available in areas other than X2Y2 and X3Y3, autofocusing can be performed there, but no autofocusing can be performed in the small areal images X2Y2, X2Y2 since no specimen is available in the small areal image of X2Y2 and a contrast of a specimen is low in the small area image of X3Y3, whereby differences in contrast level of the specimen cannot be obtained there.
Accordingly, a series of shooting of plural images cannot be continued and as a result, an image with high resolution covering the entire specimen is impossible to be attained.
Fluorescence observation is a method in which irradiation with excitation light to a specimen is effected which has been treated by fluorescent dye and an image formed by emission of weak fluorescent light from the specimen is observed.
One of features the fluorescence observation is fading. Fading of a specimen is to show over-time attenuation of fluorescent light power emitted from the specimen which is caused by irradiation with excitation light on the specimen treated by fluorescent dye.
The attenuation of the light is proportional to an intensity of excitation light.times.a irradiation time of the excitation light. Hence, an operator has to adjust the irradiation time so as to be as short as possible.
However, in order to alleviate a burden on specimen observation, in fluorescence observation, too, a demand for mounting an autofocus device on a microscope has progressively been strengthened.
An autofocus device for fluorescence observation requires the following capabilities:
1) a sensor which detects an observation image has to be of high sensitivity since fluorescent light emitted from a specimen is weak; PA1 2) a speed for achieving focus has to be high (a time required for achieving focus is short) since a time over which a specimen emits fluorescent light is short due to a fading effect; and PA1 3) high accuracy in focusing is required in order to raise reliability in inspection.
For example, a method is disclosed in Jpn. Pat. Appln. KOKAI Publication No. 54-45127, in which an integral time of an integrating light receiving element in use for detecting an observation image is controlled according to brightness of the observation image and an apparent sensitivity is thus improved.
Besides, another method is disclosed in Jpn. Pat. Appln. KOKAI Publication No. 59-154880, in which light shielding pixels or a shutter in a forward position of the shutter is used for a purpose of intake of a light image from an observation image with good accuracy and thereby noise light is removed.
In an ordinary microscope, however, environmental irradiation light and the like is mixed into an light image formed on a sensor by incident light through an objective in addition to an optical image from an observation image, that is stray light is mixed. Hence, an microscope in which a conventional autofocusing technique as it is adopted cannot be applied to fluorescence observation.
At this point, stray light will be described.
FIG. 12 is a representation showing details of optical paths of a microscope used in fluorescence observation.
Essentially, optical paths of a microscope are as follows: incident light emitted from a light source 52 of a mercury lamp and the like shown a solid line passes through a downward projection fluorescent tube 54, a specimen is irradiated with the light and a fluorescent image of a specimen S is sent to an image sensor 59 through an objective 56 as information.
However, image light which is actually supplied as input to the image sensor 59 includes light which is strayed into the image light from a different path from the normal paths.
As one example for the case, stray light is shown by a path of a broken line A in the figure. When a microscope resides in a room, irradiation light such as from fluorescent lamp is supplied into the image sensor 59 as stray light.
That is, irradiation light in the room is reflected on a stage 51 and a slide glass for the specimen S and then passes through the objective 56 to be projected on the image sensor as input. As another source of stray light, the irradiation light domes into the image sensor 59 through eyepieces 57.
Such stray light is weaker in intensity than fluorescent light from the specimen, but an autofocus device for a microscope works in a wrong manner in connection with a threshold value which is a base by a cause of the stray light as shown by the broken lines A and B in the figure since output of fluorescent light from the specimen is essentially weak. In addition, when an observation image is blurred in fluorescence observation, an observation image which is an input to the image sensor cannot be distinguished from the stray light.
When an autofocus device disclosed in Jpn. Pat. Appln. KOKAI Publication No. 54-45127 is applied to fluorescence observation, since an integral time of a sensor is adjusted in a condition where the stray light is incorporated in the course of specimen search, the integral time gets longer and amounts to a value larger than a time actually required for intake of a specimen image, which entails great reduction in a focusing speed.
In Jpn. Pat. Appln. KOKAI Publication No. 59-154880, if light shielding pixels or a shutter in a forward position of a sensor is used, removal of stray light coming through an objective is impossible as in the case of a normal specimen optical. image. Besides, since stray light is different in nature or quantity according to environmental conditions surrounding a microscope or capabilities of the microscope, stray light is hard to be removed from light including image light in advance.